Human YVH1 is a 36 kDa dual specificity phosphatase widely expressed in human tissues. Orthologues of YVH1 are conserved throughout higher eukaryotes from yeast to humans. In addition to its phosphatase domain, YVH1 members posses a C-terminal Zn-finger like domain that shows higher sequence identity than the catalytic domain. Although hYVH1 is able to complement the growth defect caused by disruption of the S. cerevisiae YVH1 gene, its physiological role in human cells is completely unknown. Data presented in this proposal demonstrates for the first time that hYVH1 is phosphorylated in vivo on serine residues. Efforts will be made to identify the hYVH1 in vivo phosphorylation sites using mass spectrometry and phosphopeptide mapping and further analyze the role of the individual phosphorylation events in regulating hYVH1 activities. Also an affinity purification-based approach in conjunction with substrate-trapping mutants and mass spectrometry will be employed to search for hYVH1 substrates and binding partners. Finally, large amounts of hYVH1 can be expressed and purified from bacteria cells. Crystallization experiments will be performed with the aim of obtaining an X-ray structure of hYVH1. It is anticipated that the structure of hYVH1 will reveal important insights into catalysis and the function of the zinc-finger binding domain.